Fig. 4.

Binding of designed inhibitors to target amyloid fibrils. (A) ELISA binding assays were used to measure inhibitor binding to fibril ends. Inhibitors with uncleaved His-tags were incubated with fibril-coated plates, then labeled with an AF₆₄₇-conjugated anti-His primary antibody. Fluorescence measurements at different concentrations of inhibitor (iTau-N shown) illustrate binding saturation in the high nanomolar regime. (B) To visualize the designed miniproteins bound to the fibril ends, inhibitors were incubated with fibrils, then labeled with 5 or 20 nm gold nanoparticles (indicated by red arrows), demonstrating a binding mode consistent with their intended design. (C–E) ELISA binding curves of iαSyn-F to αSyn fibrils (C), iTau-N to tau k18+ fibrils seeded with AD brain-derived fibrils (D), and iAβ-H to Aβ_1–42 fibrils (E). (F–H) Transmission electron micrographs of amyloid fibrils bound with miniprotein inhibitors labeled with gold nanoparticles. (F) iαSyn-E bound to αSyn fibrils. (G) iTau-N bound to tau k18+ fibrils seeded with AD brain-derived fibrils. (H) iAβ-H bound to Aβ_1–42 fibrils. In all three samples, inhibitors are observed binding primarily to the tips of the fibrils, consistent with their intended design. Additional TEM images are available in SI Appendix, Fig. 5.